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EP1080194A2 - Proteines transmembranaires humaines - Google Patents

Proteines transmembranaires humaines

Info

Publication number
EP1080194A2
EP1080194A2 EP99927108A EP99927108A EP1080194A2 EP 1080194 A2 EP1080194 A2 EP 1080194A2 EP 99927108 A EP99927108 A EP 99927108A EP 99927108 A EP99927108 A EP 99927108A EP 1080194 A2 EP1080194 A2 EP 1080194A2
Authority
EP
European Patent Office
Prior art keywords
seq
htmpn
polynucleotide
sequence
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99927108A
Other languages
German (de)
English (en)
Inventor
Y. Tom Tang
Preeti Lal
Jennifer L. Hillman
Henry Yue
Karl J. Guegler
Neil C. Corley
Olga Bandman
Chandra Patterson
Gina A. Gorgone
Matthew R. Kaser
Mariah R. Baughn
Janice Au-Young
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Incyte Pharmaceuticals Inc filed Critical Incyte Pharmaceuticals Inc
Publication of EP1080194A2 publication Critical patent/EP1080194A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • A61P31/12—Antivirals
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    • A—HUMAN NECESSITIES
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    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • TECHNICAL FIELD This invention relates to nucleic acid and amino acid sequences of human transmembrane proteins and to the use of these sequences in the diagnosis, treatment, and prevention of immune, reproductive, smooth muscle, neurological, gastrointestinal, developmental, and cell proliferative disorders.
  • Eukaryotic organisms are distinct from prokaryotes in possessing many intracellular organelle and vesicle structures. Many of the metabolic reactions which distinguish eukaryotic biochemistry from prokaryotic biochemistry take place within these structures. In particular, many cellular functions require very stringent reaction conditions, and the organelles and vesicles enable compartmentalization and isolation of reactions which might otherwise disrupt cytosolic metabolic processes.
  • the organelles include mitochondria, smooth and rough endoplasmic reticula, sarcoplasmic reticulum, and the Golgi body.
  • the vesicles include phagosomes, lysosomes, endosomes, peroxisomes, and secretory vesicles. Organelles and vesicles are bounded by single or double membranes.
  • Biological membranes are highly selective permeable barriers made up of lipid bilayer sheets composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins.
  • Membranes contain ion pumps, ion channels, and specific receptors for external stimuli which transmit biochemical signals across the membranes. These membranes also contain second messenger proteins which interact with these pumps, channels, and receptors to amplify and regulate transmission of these signals.
  • Plasma membrane proteins are divided into two groups based upon methods of protein extraction from the membrane. Extrinsic or peripheral membrane proteins can be released using extremes of ionic strength or pH, urea, or other disruptors of protein interactions. Intrinsic or integral membrane proteins are released only when the lipid bilayer of the membrane is dissolved by detergent.
  • Transmembrane proteins are characterized by an extracellular, a transmembrane, and an intracellular domain. TM domains are typically comprised of 15 to 25 hydrophobic amino acids which are predicted to adopt an ⁇ -helical conformation. TM proteins are classified as bitopic (Types I and II) proteins, which span the membrane once, and polytopic (Types III and IV) (Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247- 96) proteins which contain multiple membrane-spanning segments.
  • TM proteins that act as cell-surface receptor proteins involved in signal transduction include growth and differentiation factor receptors, and receptor-interacting proteins such as Drosophila pecanex and frizzled proteins, LIV-1 protein, NF2 protein, and GNS1/SUR4 eukaryotic integral membrane proteins.
  • TM proteins also act as transporters of ions or metabolites, such as gap junction channels (connexins), and ion channels, and as cell anchoring proteins, such as lectins, integrins, and fibronectins.
  • TM proteins are found in vesicle organelle-forming molecules, such as calveolins; or cell recognition molecules, such as cluster of differentiation (CD) antigens, glycoproteins, and mucins.
  • CD cluster of differentiation
  • membrane proteins contain amino acid sequence motifs that serve to localize proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD. NGR, and GSL sequence motifs, von Willebrand factor A (vWFA) domains, and EGF-like domains. RGD, NGR, and GSL motif-containing peptides have been used as drug delivery agents in targeted cancer treatment of tumor vasculature (Arap, W. et al. (1998) Science, 279:377-380). Membrane proteins may also contain amino acid sequence motifs that serve to interact with extracellular or intracellular molecules, such as carbohydrate recognition domains.
  • Examples of such chemical modifications to amino acid residue side chains are covalent bond formation with glycosaminoglycans, oligosaccharides, phospholipids, acetyl and palmitoyl moieties, ADP-ribose, phosphate, and sulphate groups.
  • RNA-encoding membrane proteins may have alternative splice sites which give rise to proteins encoded by the same gene but with different messenger RNA and amino acid sequences. Splice variant membrane proteins may interact with other ligand and protein isoforms. G-Protein Coupled Receptors
  • GPCR G-protein coupled receptors
  • GPCRs include receptors for biogenic amines, lipid mediators of inflammation, peptide hormones, and sensory signal mediators.
  • the structure of these highly-conserved receptors consists of seven hydrophobic transmembrane (serpentine) regions, cysteine disulfide bridges between the second and third extracellular loops, an extracellular N-terminus, and a cytoplasmic C-terminus.
  • Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. The most conserved parts of these proteins are the transmembrane regions and the first two cytoplasmic loops.
  • a conserved, acidic- Arg-aromatic residue triplet present in the second cytoplasmic loop may interact with G proteins.
  • a GPCR consensus pattern is characteristic of most proteins belonging to this superfamily (ExPASy PROSITE document PS00237; and Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego, CA, pp 2-6). Mutations and changes in transcriptional activation of GPCR-encoding genes have been associated with neurological disorders such as schizophrenia, Parkinson's disease, Alzheimer's disease, drug addiction, and feeding disorders. Scavenger Receptors
  • Macrophage scavenger receptors with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens.
  • Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N- terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C-terminal cysteine-rich domain.
  • the extracellular domain contains a short spacer domain, an ⁇ -helical coiled-coil domain, and a triple helical collagenous domain.
  • TM4SF transmembrane 4 superfamily
  • TM4SF transmembrane 4 superfamily
  • TM4SF is a multigene family encoding type III integral membrane proteins (Wright, M.D. and Tomlinson, M.G. (1994) Immunol. Today 15:588).
  • TM4SF is comprised of membrane proteins which traverse the cell membrane four times.
  • Members of the TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tumor-associated antigens, and surface proteins of the schistosome parasites (Jankowski, S.A. (1994) Oncogene 9:1205-1211).
  • Members of the TM4SF share about 25-30% amino acid sequence identity with one another.
  • TM4SF members have been implicated in signal transduction, control of cell adhesion, regulation of cell growth and proliferation, including development and oncogenesis. and cell motility. including tumor cell metastasis. Expression of TM4SF proteins is associated with a variety of tumors and the level of expression may be altered when cells are growing or activated.
  • Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tumor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. Cancer 61 : 706-715; Liu, E. et al. (1992) Oncogene 7: 1027-1032).
  • Ion channels Ion channels are found in the plasma membranes of virtually every cell in the body. For example, chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ions across epithelial membranes.
  • chloride channels When present in intracellular membranes of the Golgi apparatus and endocytic vesicles, chloride channels also regulate organelle pH (see, e.g., Greger, R. (1988) Annu. Rev. Physiol. 50: 111-122). Electrophysiological and pharmacological properties of chloride channels, including ion conductance, current- voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
  • Many channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all of which regulate ion channel activity in cells.
  • Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, and skeletal muscle.
  • Proton pumps Proton ATPases are a large class of membrane proteins that use the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane.
  • the resultant gradient may be used to transport other ions across the membrane (Na + , K + , or Cl " ) or to maintain organelle pH.
  • Proton ATPases are further subdivided into the mitochondrial F-ATPases, the plasma membrane ATPases, and the vacuolar ATPases.
  • the vacuolar ATPases establish and maintain an acidic pH within various vesicles involved in the processes of endocytosis and exocytosis (Mellman, I. et al. (1986) Ann. Rev. Biochem. 55:663-700).
  • Proton-coupled, 12 membrane-spanning domain transporters such as PEPT 1 and PEPT 2 are responsible for gastrointestinal absorption and for renal reabsorbtion of peptides using an electrochemical H + gradient as the driving force.
  • Another type of peptide transporter, the TAP transporter is a heterodimer consisting of TAP 1 and TAP 2 and is associated with antigen processing. Peptide antigens are transported across the membrane of the endoplasmic reticulum by TAP so they can be expressed on the cell surface in association with MHC molecules.
  • Each TAP protein consists of multiple hydrophobic membrane spanning segments and a highly conserved ATP-binding cassette (Boll, M. et al. (1996) Proc. Natl. Acad. Sci.
  • Pathogenic microorganisms such as herpes simplex virus, may encode inhibitors of TAP-mediated peptide transport in order to evade immune surveillance (Marusina, K. and Manaco, JJ. (1996) Curr. Opin. Hematol. 3:19-26). ABC Transporters
  • ABC transporters also called the "traffic ATPases”
  • the ATP-binding cassette (ABC) transporters comprise a superfamily of membrane proteins that mediate transport and channel functions in prokaryotes and eukaryotes (Higgins, C.F. (1992) Annu. Rev. Cell Biol. 8:67-113).
  • ABC proteins share a similar overall structure and significant sequence homology. All ABC proteins contain a conserved domain of approximately two hundred amino acid residues which includes one or more nucleotide binding domains.
  • ABC transporter genes are associated with various disorders, such as hyperbilirubinemia II/Dubin-Johnson syndrome, recessive Stargardt's disease, X-linked adrenoluekodystrophy, multidrug resistance, celiac disease, and cystic fibrosis.
  • Intercellular communication is essential for the development and survival of multicellular organisms.
  • Cells communicate with one another through the secretion and uptake of protein signaling molecules.
  • the uptake of proteins into the cell is achieved by endocytosis. in which the interaction of signaling molecules with the plasma membrane surface, often via binding to specific receptors, results in the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol.
  • the secretion of proteins from the cell is achieved by exocytosis, in which molecules inside of the cell are packaged into membrane-bound transport vesicles derived from the trans- Golgi network. These vesicles fuse with the plasma membrane and release their contents into the surrounding extracellular space. Endocytosis and exocytosis result in the removal and addition of plasma membrane components and the recycling of these components is essential to maintain the integrity, identity, and functionality of both the plasma membrane and internal membrane-bound compartments.
  • Lysosomes are the site of degradation of intracellular material during autophagy and of extracellular molecules following endocytosis. Lysosomal enzymes are packaged into vesicles which bud from the tr ⁇ ra-Golgi network. These vesicles fuse with endosomes to form the mature lysosome in which hydrolytic digestion of endocytosed material occurs. Lysosomes can fuse with autophagosomes to form a unique compartment in which the degradation of organelles and other intracellular components occurs.
  • Protein sorting by transport vesicles has important consequences for a variety of physiological processes including cell surface growth, the biogenesis of distinct intracellular organelles, endocytosis, and the controlled secretion of hormones and neurotransmitters (Rothman, J.E. and Wieland, F.T. (1996) Science 272:227-234).
  • neurodegenerative disorders and other neuronal pathologies are associated with biochemical flaws during endosomal protein sorting or endosomal biogenesis (Mayer RJ. et al. (1996) Adv. Exp. Med. Biol. 389:261-269).
  • Peroxisomes are organelles independent from the secretory pathway.
  • Peroxisomes are the site of many peroxide-generating oxidative reactions in the cell.
  • Peroxisomes are unique among eukaryotic organelles in that their size, number, and enzyme content vary depending upon organism, cell type, and metabolic needs.
  • the majority of peroxisome- associated proteins are membrane-bound or are found proximal to the cytosolic or the lumenal side of the peroxisome membrane (Waterham, H.R. and Cregg, J.M. (1997) BioEssays 19:57-66).
  • the normal functioning of the eukaryotic cell requires that all newly synthesized proteins be correctly folded, modified, and delivered to specific intra- and extracellular sites.
  • Newly synthesized membrane and secretory proteins enter a cellular sorting and distribution network during or immediately after synthesis and are routed to specific locations inside and outside of the cell.
  • the initial compartment in this process is the endoplasmic reticulum (ER) where proteins undergo modifications such as glycosylation, disulfide bond formation, and assembly into oligomers.
  • ER endoplasmic reticulum
  • the modified proteins are then transported through a series of membrane-bound compartments which include the various cisternae of the Golgi complex, where further carbohydrate modifications occur.
  • Transport between compartments occurs by means of vesicles that bud and fuse in a manner specific to the type of protein being transported.
  • proteins Once within the secretory pathway, proteins do not have to cross a membrane to reach the cell surface. Although the majority of proteins processed through the ER are transported out of the organelle, some are retained.
  • the signal for retention in the ER in mammalian cells consists of the tetrapeptide sequence, KDEL, located at the carboxyl terminus of proteins (Munro, S. (1986) Cell 46:291-300). Proteins containing this sequence leave the ER but 5 are quickly retrieved from the early Golgi cisternae and returned to the ER, while proteins lacking this signal continue through the secretory pathway.
  • ⁇ -a yloid precursor protein involves the putative vesicle transport protein presenilin, and may play a role in early- onset Alzheimer's disease (Levy-Lahad. E. et al. (1995) Science 269:973-977).
  • the mitochondrial electron transport (or respiratory) chain is a series of three enzyme complexes in the mitochondrial membrane that is responsible for the transport of electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of 0 ATP (oxidative phosphorylation). ATP then provides the primary source of energy for driving the many energy-requiring reactions of a cell.
  • B-cell response to antigens is an essential component of the normal immune system.
  • Mature B cells recognize foreign 0 antigens through B cell receptors (BCR) which are membrane-bound, specific antibodies that bind foreign antigens.
  • BCR B cell receptors
  • the antigen/receptor complex is internalized and the antigen is proteolytically processed.
  • the BCR, BCR-associated proteins, and Tcell response are all required.
  • Proteolytic fragments of the antigen are complexed with major histocompatability complex-II (MHCII) molecules on the surface of the B cells where the complex can be recognized by T cells.
  • MHCII major histocompatability complex-I
  • lymphoid cells present antigens in association with MHCI molecules to T cells.
  • T cells recognize and are activated by the MHCI-antigen complex through interactions with the T cell receptor/CD3 complex, a T cell-surface multimeric protein located in the plasma membrane.
  • T cells activated by antigen presentation secrete a variety of lymphokines that induce B cell maturation and T cell proliferation and activate macrophages, which kill target cells.
  • Leukocytes have a fundamental role in the inflammatory and immune response and include monocytes/macrophages, mast cells, polymorphonucleoleukocytes, natural killer cells, neutrophils, eosinophils, basophils, and myeloid precursors.
  • Leukocyte membrane proteins include members of the CD antigens, N-CAM, I-CAM, human leukocyte antigen (HLA) class I and HLA class II gene products, immunoglobulins, immunoglobulin receptors, complement, complement receptors, interferons, interferon receptors, interleukin receptors, and chemokine receptors.
  • Abnormal lymphocyte and leukocyte activity has been associated with acute disorders, such as AIDS, immune hypersensitivity, leukemias, leukopenia, systemic lupus, granulomatous disease, and eosinophilia.
  • Apoptosis-Associated Membrane Proteins include AIDS, immune hypersensitivity, leukemias, leukopenia, systemic lupus, granulomatous disease, and eosinophilia.
  • a variety of ligands, receptors, enzymes, tumor suppressors, viral gene products, pharmacological agents, and inorganic ions have important positive or negative roles in regulating and implementing the apoptotic destruction of a cell. Although some specific components of the apoptotic pathway have been identified and characterized, many interactions between the proteins involved are undefined, leaving major aspects of the pathway unknown.
  • Tumorgenesis is associated with the activation of oncogenes which are derived from normal cellular genes. These oncogenes encode oncoproteins which are capable of converting normal cells into malignant cells. Some oncoproteins are mutant isoforms of the normal protein and other oncoproteins are abnormally expressed with respect to location or level of expression. The latter category of oncoprotein causes cancer by altering transcriptional control of cell proliferation.
  • Five classes of oncoproteins are known to affect the cell cycle controls. These classes include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell- cycle control proteins. These proteins include those which are modified by glycosylation, phosphorylation, glycosaminoglycan attachment, sulphation, and lipidation.
  • Modulation of factors which act in the coordination of the human cell division cycle may provide an important means to reduce tumorgenesis.
  • An example of the metastasis-associated proteins is the lysosomal membrane glycoprotein P2B/LAMP-1 which is also expressed in normal tissues.
  • P2B/LAMP-1 which is also expressed in normal tissues.
  • mammalian proteins homologous to the plant pathogenesis- related proteins have been identified in hyperplastic glioma. (Murphy, EN. et al. (1995) Gene 159:131-135.)
  • HTMPN substantially purified polypeptides, human transmembrane proteins, referred to collectively as "HTMPN” and individually as “HTMPN- 1", “HTMPN-2", “HTMPN-3", “HTMPN-4", “HTMPN-5”, “HTMPN-6", “HTMPN-7”, “HTMPN-8", “HTMPN-9”, “HTMPN-10”, “HTMPN-11”, “HTMPN-12”, “HTMPN-13”, “HTMPN-14”, “HTMPN-15”, “HTMPN-16", “HTMPN-17”, “HTMPN-18”, “HTMPN- 19", “HTMPN-20”, “HTMPN-21”, “HTMPN-22”, “HTMPN-23”, “HTMPN-24”, “HTMPN-25”, “HTMPN-26", “HTMPN-27”, “HTMPN-28", “HTMPN-29”, “HTMPN- 30”, “HTMPN-31", “HTMPN-32", “HTMPN-33”, “HTMPN-34”, “HTMPN-
  • the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2,
  • SEQ ID NO:3 SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25.
  • SEQ ID NO:26 SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, 0 SEQ ID NO:30, SEQ ID NO:31 , SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID 5 NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61
  • the invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1-79, and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-79, and fragments thereof.
  • the invention also includes an isolated and purified polynucleotide variant having at least 90%) polynucleotide sequence identity to the polynucleotide encoding the polypeptide 5 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 79, and fragments thereof.
  • the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-
  • the invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1-79, and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide comprising a
  • polynucleotide sequence selected from the group consisting of SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID 0 NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:l 10, SEQ ID NO:l 11, SEQ ID NO:
  • the invention further provides an isolated and purified polynucleotide variant having at least 90%) polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO:80-158, and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:80-158, and fragments thereof.
  • the invention also provides a method for detecting a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide in the sample.
  • the method further comprises amplifying the polynucleotide prior to hybridization.
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-79, and fragments thereof.
  • the expression vector is contained within a host cell.
  • the invention also provides a method for producing a polypeptide, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-79, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
  • the invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO: 1 -79, and fragments thereof.
  • the invention also provides a purified agonist and a purified antagonist to the polypeptide.
  • the invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of HTMPN, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1-79, and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
  • the invention also provides a method for treating or preventing a disorder associated with increased expression or activity of HTMPN, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-79, and fragments thereof.
  • clone ID NOs clone identification numbers (clone ID)
  • cDNA libraries clone fragments used to assemble full-length sequences encoding HTMPN.
  • Table 2 shows features of each polypeptide sequence including predicted transmembrane sequences, potential motifs, homologous sequences, and methods and algorithms used for identification of HTMPN.
  • Table 3 shows the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis, diseases, disorders, or conditions associated with these tissues, and the vector into which each cDNA was cloned.
  • Table 4 describes the tissues used to construct the cDNA libraries from which Incyte cDNA clones encoding HTMPN were isolated.
  • Table 5 shows the programs, their descriptions, references, and threshold parameters used to analyze HTMPN.
  • HTMPN refers to the amino acid sequences of substantially purified HTMPN obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which, when bound to HTMPN, increases or prolongs the duration of the effect of HTMPN.
  • Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of HTMPN.
  • allelic variant is an alternative form of the gene encoding HTMPN. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding HTMPN include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polynucleotide the same as HTMPN or a polypeptide with at least one functional characteristic of HTMPN. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HTMPN, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HTMPN.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HTMPN.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of HTMPN is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
  • amino acid or amino acid sequence refer to an oligopeptide, peptide, polypeptide. or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules.
  • fragments refer to fragments of HTMPN which are preferably at least 5 to about 15 amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of HTMPN.
  • amino acid sequence is recited to refer to an amino acid sequence of a naturally occurring protein molecule
  • amino acid sequence and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
  • Antagonist refers to a molecule which, when bound to HTMPN, decreases the amount or the duration of the effect of the biological or immunological activity of HTMPN.
  • Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of HTMPN.
  • antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
  • Antibodies that bind HTMPN polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition containing a nucleic acid sequence which is complementary to the "sense” strand of a specific nucleic acid sequence. Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation “negative” can refer to the antisense strand, and the designation “positive” can refer to the sense strand.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant, or synthetic HTMPN, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementarity refers to the natural binding of polynucleotides by base pairing. For example, the sequence “5' A-G-T 3"' bonds to the complementary sequence "3' T-C-A 5'.”
  • Complementarity between two single-stranded molecules may be "partial,” such that only some of the nucleic acids bind, or it may be "complete,” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.
  • composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding HTMPN or fragments of HTMPN may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW Fragment Assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
  • correlates with expression of a polynucleotide indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding HTMPN, by northern analysis is indicative of the presence of nucleic acids encoding HTMPN in a sample, and thereby correlates with expression of the transcript from the polynucleotide encoding HTMPN.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • similarity refers to a degree of complementarity. There may be partial similarity or complete similarity. The word “identity” may substitute for the word “similarity.”
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency.
  • a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency.
  • Percent identity refers to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR, Madison WI) which creates alignments between two or more sequences according to methods selected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned pairwise and then in groups.
  • the percentage similarity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods known in the art, e.g., by varying hybridization conditions.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
  • humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion or “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • factors e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • microarray refers to an arrangement of distinct polynucleotides on a substrate.
  • element or “array element” in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
  • modulate refers to a change in the activity of HTMPN. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HTMPN.
  • nucleic acid or “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof.
  • RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • fragment refers to those nucleic acid sequences which, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP- binding site, of the full-length polypeptide.
  • operably associated or “operably linked” refer to functionally related nucleic acid sequences.
  • a promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polypeptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to operator sequences that control expression of the polypeptide.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
  • Oligonucleotide is substantially equivalent to the terms “amplimer,” “primer.” “oligomer,” and “probe,” as these terms are commonly defined in the art.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • sample is used in its broadest sense.
  • a sample suspected of containing nucleic acids encoding HTMPN, or fragments thereof, or HTMPN itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • specific binding or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule.
  • an antibody is specific for epitope "A”
  • the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • stringent conditions refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides.
  • Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions well known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • Transformation describes a process by which exogenous DNA enters and changes a recipient cell.
  • Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell.
  • the method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • the term "transformed" cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a “variant" of HTMPN polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative” changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
  • variants when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to HTMPN. This definition may also include, for example, "allelic” (as defined above), “splice,” “species,” or “polymorphic” variants.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or an absence of domains.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population, a disease state. or a propensity for a disease state.
  • the invention is based on the discovery of new human transmembrane proteins (HTMPN), the polynucleotides encoding HTMPN, and the use of these compositions for the diagnosis, treatment, or prevention of immune, reproductive, smooth muscle, neurological, gastrointestinal, developmental, and cell proliferative disorders.
  • HTMPN human transmembrane proteins
  • Table 1 lists the Incyte Clones used to derive full length nucleotide sequences encoding HTMPN. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the amino acid and nucleic acid sequences, respectively. Column 3 shows the Clone ID of the Incyte Clone in which nucleic acids encoding each HTMPN were identified, and column 4, the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones, their corresponding cDNA libraries, and shotgun sequences. The clones and shotgun sequences are part of the consensus nucleotide sequence of each HTMPN and are useful as fragments in hybridization technologies.
  • column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide; column 3, potential phosphorylation sites; column 4, potential glycosylation sites; column 5, the amino acid residues comprising signature sequences and motifs; column 6, the identity of each protein; and column 7, analytical methods used to identify each protein through sequence homology and protein motifs.
  • Hidden Markov Model analysis indicates the presence of one or more potential transmembrane motifs in each of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO: 66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO: 79; as well as the presence of one or more potential signal peptide motifs in each of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:77, and SEQ ID NO:79.
  • Motifs analysis indicates the presence of a potential ATP/GTP binding site in SEQ ID NO:68, a potential calcium-binding site also in SEQ ID NO:68, a potential leucine zipper gene regulatory motif in each of SEQ ID NO:68 and SEQ ID NO:73; and a potential microbody (single-membraned organelle) targeting signal site in SEQ ID NO:78.
  • BLOCKS analysis indicates the presence of two potential PMP-22 integral membrane glycoprotein motifs and a trehalase motif, all in SEQ ID NO: 77, as well as a potential protein-splicing motif in SEQ ID NO:66.
  • PRINTS analysis indicates the presence of a potential G-protein coupled receptor motif in SEQ ID NO:79.
  • the columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding HTMPN.
  • the first column of Table 3 lists the nucleotide sequence identifiers.
  • the second column lists tissue categories which express HTMPN as a fraction of total tissue categories expressing HTMPN.
  • the third column lists the diseases, disorders, or conditions associated with those tissues expressing HTMPN.
  • the fourth column lists the vectors used to subclone the cDNA library.
  • HTMPN in tissue involved in inflammation and the immune response and with cell proliferative conditions including cancer, and in reproductive, gastrointestinal, fetal, smooth muscle, cardiovascular, urologic, endocrine, developmental, and nervous tissue.
  • the following fragments of the nucleotide sequences encoding HTMPN are useful in hybridization or amplification technologies to identify SEQ ID NO: 121 -158 and to distinguish between SEQ ID NO: 121 -158 and related polynucleotide sequences.
  • the useful fragments are the fragment of SEQ ID NO : 121 from about nucleotide 151 to about nucleotide 189; the fragment of SEQ ID NO: 122 from about nucleotide 280 to about nucleotide 318; the fragment of SEQ ID NO: 123 from about nucleotide 505 to about nucleotide 558; the fragments of SEQ ID NO:124 from about nucleotide 1 to about nucleotide 21 and from about nucleotide 694 to about nucleotide 720; the fragment of SEQ ID NO:125 from about nucleotide 331 to about nucleotide 378; the fragment of SEQ ID NO: 126 from about nucleotide 1012 to about nucleotide 1047; the fragment of SEQ ID NO:127 from about nucleotide 1070 to about nucleotide 1106; the fragment of SEQ ID NO: 128 from about nucleotide 133 to about nucleotide 186; the
  • the invention also encompasses HTMPN variants.
  • a preferred HTMPN variant is one which has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% amino acid sequence identity to the HTMPN amino acid sequence, and which contains at least one functional or structural characteristic of HTMPN.
  • the invention also encompasses polynucleotides which encode HTMPN.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:80-158, which encodes HTMPN.
  • the invention also encompasses a variant of a polynucleotide sequence encoding HTMPN.
  • a variant polynucleotide sequence will have at least about 80%o, more preferably at least about 90%), and most preferably at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding HTMPN.
  • a particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:80-158 which has at least about 80%, more preferably at least about 90%>, and most preferably at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:80-158.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HTMPN.
  • nucleotide sequences which encode HTMPN and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HTMPN under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HTMPN or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode HTMPN and HTMPN derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a sequence encoding HTMPN or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:80-158 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency
  • hybridization can be obtained in the presence of at least about 35%) formamide, and most preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and
  • hybridization will occur at 30°C in 750 mM NaCl. 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
  • ssDNA 100 ⁇ g/ml denatured salmon sperm DNA
  • hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS. 50 % formamide. and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • washing steps which follow hybridization can also vary in stringency.
  • 25 stringency conditions can be defined by salt concentration and by temperature.
  • wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • wash steps will ordinarily include temperature of at least about 25 °C, more preferably of at least about 42°C, and most preferably of at least about 68°C.
  • wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1 % SDS.
  • wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1%> SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life
  • sequence preparation is automated with machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno NV), Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown MA) and the ABI CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI 373 or 377 DNA sequencing systems (Perkin-Elmer) or the MEGABACE 1000 DNA sequencing system (Molecular Dynamics. Sunnyvale CA). The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853.)
  • the nucleic acid sequences encoding HTMPN may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector.
  • PCR Methods Applic. 2:318-322 Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences.
  • a third method, capture PCR involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic.
  • primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotide sequences or fragments thereof which encode HTMPN may be cloned in recombinant DNA molecules that direct expression of HTMPN, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express HTMPN.
  • the nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter HTMPN-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR 5 reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
  • sequences encoding HTMPN may be synthesized, in
  • HTMPN itself or a fragment thereof may be synthesized using chemical methods.
  • peptide synthesis can be performed using various solid-phase techniques.
  • Automated synthesis may be achieved using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid sequence of HTMPN, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • the peptide may be substantially purified by preparative high performance liquid 0 chromatography. (See. e.g, Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol.
  • composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties. WH Freeman, New York NY.)
  • the nucleotide sequences 5 encoding HTMPN or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding 0 HTMPN. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HTMPN. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding HTMPN. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • the invention is not limited by the host cell employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HTMPN.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding HTMPN can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or pSPORTl plasmid (Life Technologies). Ligation of sequences encoding HTMPN into the vector's multiple cloning site disrupts the lacL gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of HTMPN may be used.
  • vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of HTMPN.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of HTMPN. Transcription of sequences encoding HTMPN may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York NY, pp. 191-196.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding HTMPN may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses HTMPN in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer
  • RSV Rous sarcoma virus
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, JJ. et al. (1997) Nat Genet. 15:345-355.)
  • sequences encoding HTMPN can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ⁇ or apr ⁇ cells, respectively. (See, e.g., Wigler. M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite. antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides.
  • neomycin and G-418 and als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites.
  • trpB and hisD which alter cellular requirements for metabolites.
  • Visible markers e.g., anmocyanins, green fluorescent proteins (GFP; Clontech), ⁇ glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131.) Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • GFP green fluorescent proteins
  • transformed cells containing sequences encoding HTMPN can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding HTMPN under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding HTMPN and that express HTMPN may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations. PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of HTMPN using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HTMPN is preferred, but a competitive binding assay may be employed.
  • assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual. APS Press, St Paul MN, Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols. Humana Press, Totowa NJ).
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HTMPN include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding HTMPN, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3.
  • reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding HTMPN may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode HTMPN may be designed to contain signal sequences which direct secretion of HTMPN through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation. phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC, Bethesda MD) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • natural, modified, or recombinant nucleic acid sequences encoding HTMPN may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric HTMPN protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HTMPN activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, giutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized giutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the HTMPN encoding sequence and the heterologous protein sequence, so that HTMPN may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled HTMPN may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 35 S-methionine.
  • HTMPN fragments of HTMPN may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra. pp. 55-60.) Protein synthesis may be performed by manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Various fragments of HTMPN may be synthesized separately and then combined to produce the full length molecule. THERAPEUTICS Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of HTMPN and human transmembrane proteins.
  • HTMPN is closely associated with tissue involved in inflammation and the immune response and with cell proliferative conditions including cancer, and in reproductive, gastrointestinal, fetal, smooth muscle, cardiovascular, developmental, and nervous tissue. Therefore, HTMPN appears to play a role in immune, reproductive, smooth muscle, neurological, gastrointestinal, developmental, and cell proliferative disorders.
  • immune, reproductive, smooth muscle, neurological, gastrointestinal, developmental, and cell proliferative disorders associated with increased HTMPN expression or activity it is desirable to decrease the expression or activity of HTMPN.
  • HTMPN In the treatment of the above conditions associated with decreased HTMPN expression or activity, it is desirable to increase the expression or activity of HTMPN.
  • HTMPN or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HTMPN.
  • disorders include, but are not limited to, an immune disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomer
  • an immune disorder such
  • a reproductive disorder such as a a disorder of prolactin production
  • infertility including tubal disease, ovulatory defects, and endometriosis: a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis
  • cancer of the breast, fibrocystic breast disease, and galactorrhea disruptions of spermatogenesis, abnormal sperm physiology, cancer of the testis, cancer of the prostate, benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia
  • a smooth muscle disorder such as angina, ana
  • esophagitis esophageal spasm
  • esophageal stricture esophageal carcinoma
  • dyspepsia indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis. gastroparesis.
  • antral or pyloric edema abdominal angina, pyrosis, gastroenteritis, intestinal obstruction, infections of the intestinal tract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma, biliary tract disease, hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis, Crohn's disease, Whipple's disease, Mallory- Weiss syndrome, colonic carcinoma, colonic obstruction, irritable bowel syndrome, short bowel syndrome, diarrhea, constipation, gastrointestinal hemorrhage, and acquired immunodeficiency syndrome (AIDS) enteropathy, cirrhosis, jaundice, cholestasis, hereditary hyperbilirubinemia, hepatic encephalopathy, hepatorenal syndrome, hepatitis, hepatic steatosis, hemochromatosis, Wilson's disease, ⁇ r antitry
  • a pharmaceutical composition comprising a substantially purified HTMPN in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HTMPN including, but not limited to, those provided above.
  • an agonist which modulates the activity of HTMPN may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HTMPN including, but not limited to, those listed above.
  • an antagonist of HTMPN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HTMPN. Examples of such disorders include, but are not limited to, those described above.
  • an antibody which specifically binds HTMPN may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HTMPN.
  • a vector expressing the complement of the polynucleotide encoding HTMPN may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HTMPN including, but not limited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of HTMPN may be produced using methods which are generally known in the art. In particular, purified HTMPN may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HTMPN.
  • Antibodies to HTMPN may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HTMPN or with any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially preferable.
  • the oligopeptides, peptides, or fragments used to induce antibodies to HTMPN have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HTMPN amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to HTMPN may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • the hybridoma technique the human B-cell hybridoma technique
  • EBV-hybridoma technique See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, RJ. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.
  • chimeric antibodies such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity.
  • techniques developed for the production of single chain antibodies may be adapted, using methods known in the art, to produce HTMPN-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for HTMPN may also be generated.
  • fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • K j an association constant
  • High-affinity antibody preparations with K a ranging from about IO 9 to IO 12 L/mole are preferred for use in immunoassays in which the HTMPN-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K ranging from about IO 6 to 10 7 L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of HTMPN, preferably in active form, from the antibody (Catty, D. ( 1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington, DC; Liddell. J. E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies. John Wiley & Sons. New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is preferred for use in procedures requiring precipitation of HTMPN-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)
  • the polynucleotides encoding HTMPN may be used for therapeutic purposes.
  • the complement of the polynucleotide encoding HTMPN may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding HTMPN.
  • complementary molecules or fragments may be used to modulate HTMPN activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HTMPN.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding HTMPN. (See, e.g., Sambrook, supra: Ausubel, 1995, supra.)
  • Genes encoding HTMPN can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding HTMPN. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding HTMPN.
  • Oligonucleotides derived from the transcription initiation site e.g., between about positions - 10 and +10 from the start site, are preferred.
  • inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HTMPN.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HTMPN. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half-life.
  • flanking sequences at the 5' and/or 3' ends of the molecule Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See. e.g., Goldman, C.K. et al. (1997) Nature Biotechnology 15:462-466.)
  • Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of HTMPN, antibodies to HTMPN, and mimetics, agonists, antagonists, or inhibitors of HTMPN.
  • compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • a stabilizing compound such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial. intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal. intranasal, enteral, topical, sublingual, or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees. capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2%> to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency, and method of administration.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example HTMPN or fragments thereof, antibodies of HTMPN, and agonists, antagonists or inhibitors of HTMPN, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the LD 5O ED 50 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind HTMPN may be used for the diagnosis of disorders characterized by expression of HTMPN, or in assays to monitor patients being treated with HTMPN or agonists, antagonists, or inhibitors of HTMPN.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for HTMPN include methods which utilize the antibody and a label to detect HTMPN in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • HTMPN human immunosorbent protein
  • ELISAs ELISAs
  • RIAs RIAs
  • FACS fluorescence-activated protein kinase-activated protein kinase-activated protein kinase-activated protein kinase-activated protein kinase-activated protein kinase-activated protein kinase-activated protein
  • the polynucleotides encoding HTMPN may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HTMPN may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of HTMPN. and to monitor regulation of HTMPN levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HTMPN or closely related molecules may be used to identify nucleic acid sequences which encode HTMPN.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding HTMPN, allelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably have at least 50%) sequence identity to any of the HTMPN encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 80- 158 or from genomic sequences including promoters, enhancers, and introns of the HTMPN gene.
  • Means for producing specific hybridization probes for DNAs encoding HTMPN include the cloning of polynucleotide sequences encoding HTMPN or HTMPN derivatives into vectors for the production of mRNA probes. Such vectors are known in the art.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding HTMPN may be used for the diagnosis of disorders associated with expression of HTMPN.
  • disorders include, but are not limited to, an immune disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis. amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis. autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis.
  • AIDS acquired immunodeficiency syndrome
  • Addison's disease adult respiratory distress syndrome
  • allergies ankylosing spondylitis.
  • amyloidosis anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis.
  • psoriasis Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a reproductive disorder such as a a disorder of prolactin production; infertility, including tubal disease, ovulatory defects, and endometriosis: a disruption of the estrous cycle, a disruption of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of
  • cancer of the prostate benign prostatic hyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma of the male breast, and gynecomastia; a smooth muscle disorder such as angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing ' s syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, and pheochromocytoma.
  • a smooth muscle disorder such as angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock, Cushing ' s syndrome, hypertension, hypoglycemia, myocardial infarction, migraine, and pheochromocytoma.
  • myopathies including cardiomyopathy, encephalopathy, epilepsy, Kearns-Sayre syndrome, lactic acidosis, myoclonic disorder, and ophthalmoplegia; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer ' s disease, Pick ' s disease, Huntington ' s disease, dementia, Parkinson ' s disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias.
  • a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer ' s disease, Pick ' s disease, Huntington ' s disease, dementia, Parkinson ' s disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retin
  • multiple sclerosis and other demyelinating diseases bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease; prion diseases including kuru, Creutzfeldt- Jakob disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic myopathies; myas
  • hepatic vein thrombosis veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumors including nodular hyperplasias.
  • adenomas, and carcinomas a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid,
  • the polynucleotide sequences encoding HTMPN may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin. and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered HTMPN expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding HTMPN may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding HTMPN may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
  • the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HTMPN in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding HTMPN, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • oligonucleotides designed from the sequences encoding HTMPN may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding HTMPN, or a fragment of a polynucleotide complementary to the polynucleotide encoding HTMPN, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of HTMPN include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
  • radiolabeling or biotinylating nucleotides include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves.
  • the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • Microarrays may be prepared, used, and analyzed using methods known in the art.
  • nucleic acid sequences encoding HTMPN may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) site. Correlation between the location of the gene encoding HTMPN on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 1 lq22-23.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • HTMPN, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between HTMPN and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with HTMPN, or fragments thereof, and washed. Bound HTMPN is then detected by methods well known in the art. Purified HTMPN can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the nucleotide sequences which encode HTMPN may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • RNA was purchased from Clontech or isolated from tissues described in Table 4. Some tissues were homogenized and lysed in guanidinium isothiocyanate. while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • poly(A- ) RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Valencia CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6). Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • cDNA was size-selected (300-1000 bp) using SEPHACRYL SI 000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis.
  • cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), pSPORTl plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
  • PBLUESCRIPT plasmid e.g., PBLUESCRIPT plasmid (Stratagene), pSPORTl plasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
  • Recombinant plasmids were transformed into competent E. coli cells including XL 1 -Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Life Technologies. II. Isolation of cDNA Clones Plasmids were recovered from host cells by in vivo excision, using the UNIZAP vector system (Stratagene) or cell lysis.
  • Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the REAL Prep 96 plasmid kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4°C Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B.
  • the cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin- Elmer) or the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermal cyclers (MJ Research).
  • the cDNAs were sequenced using the ABI PRISM 373 or 377 sequencing systems (Perkin- Elmer) and standard ABI protocols, base calling software, and kits.
  • cDNAs were sequenced using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics).
  • the cDNAs were amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In yet another alternative.
  • cDNAs were sequenced using solutions and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were determined using standard methods (reviewed in Ausubel. 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V.
  • the polynucleotide sequences derived from cDNA, extension, and shotgun sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art.
  • Table 5 summarizes the software programs, descriptions, references, and threshold parameters used. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides a brief description thereof, the third column presents the references which are incorporated by reference herein, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the probability the greater the homology). Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
  • the polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation, using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • HMM Hidden Markov Model
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.)
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • the results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding HTMPN occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic.
  • the disease/condition categories included cancer, inflammation trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
  • V. Extension of HTMPN Encoding Polynucleotides Full length nucleic acid sequences of SEQ ID NOs:80-120 were produced by extension of the component fragments described in Table 1, column 5, using oligonucleotide primers based on these fragments. For each nucleic acid sequence, one primer was synthesized to initiate extension of an antisense polynucleotide, and the other was synthesized to initiate extension of a sense polynucleotide.
  • Primers were used to facilitate the extension of the known sequence "outward" generating amplicons containing new unknown nucleotide sequence for the region of interest.
  • the initial primers were designed from the cDNA using OLIGOTM 4.06 (National Biosciences, Madison, MN), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68°C to about 72°C Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.
  • PCRTM kit (The Perkin-Elmer Corp., Norwalk, CT) and thoroughly mixing the enzyme and reaction mix. PCR was performed using the PTC-200 thermal cycler (MJ Research,
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 5 65° C for 1 min
  • Step 7 Repeat steps 4 through 6 for an additional 15 cycles
  • Step 8 94° C for 15 sec
  • Step 1 Repeat steps 8 through 10 for an additional 12 cycles
  • coli mixture was plated on Luria Bertani (LB) agar (See, e.g., Sambrook, supra. Appendix A, p. 1) containing carbenicillin (2x carb). The following day, several colonies were randomly picked from each plate and cultured in 150 ⁇ l of liquid LB/2x carb medium placed in an individual well of an appropriate commercially-available sterile 96-well microtiter plate. The following day, 5 ⁇ l of each overnight culture was transferred into a non-sterile 96-well plate and, after dilution 1 :10 with water, 5 ⁇ l from each sample was transferred into a PCR array.
  • LB Luria Bertani
  • PCR amplification For PCR amplification, 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer, and one or both of the gene specific primers used for the extension reaction were added to each well. Amplification was performed using the following conditions:
  • Step 2 94° C for 20 sec
  • Step 3 55° C for 30 sec
  • Step 4 72° C for 90 sec
  • Step 5 Repeat steps 2 through 4 for an additional 29 cycles
  • Step 6 72° C for 180 sec Step 7 4° C (and holding)
  • the full length nucleic acid sequences of SEQ ID NO: 121-158 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment.
  • One primer was synthesized to initiate 5' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment.
  • the initial primers were designed using OLIGO 4.06 software (National
  • the reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and ⁇ -mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min: Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3.
  • Step 6 68°C, 5 min
  • Step 7 storage at 4°C
  • the parameters for primer pair T7 and SK+ were as follows: Step 1 : 94 °C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min; Step 7: storage at 4°C
  • the concentration of DNA in each well was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transferred to 384- well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to religation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media.
  • the cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1 : 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above.
  • Hybridization probes derived from SEQ ID NO:80-158 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the- art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer. 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II. Eco Rl, Pst I, Xbal, or Pvu II (DuPont NEN).
  • a chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate.
  • An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
  • nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
  • Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena. M. et al.
  • Sequences complementary to the HTMPN-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HTMPN. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of HTMPN. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the HTMPN-encoding transcript.
  • HTMPN HTMPN expression and purification of HTMPN is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express HTMPN upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).
  • HTMPN in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Auto raphica calif ornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Auto raphica calif ornica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding HTMPN by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases.
  • HTMPN is synthesized as a fusion protein with, e.g., giutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST a 26-kilodalton enzyme from Schistosoma iaponicum. enables the purification of fusion proteins on immobilized giutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech).
  • the GST moiety can be proteolytically cleaved from HTMPN at specifically engineered sites.
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch 10 and 16). Purified HTMPN obtained by these methods can be used directly in the following activity assay. X. Demonstration of HTMPN Activity
  • HTMPN is identified as a new member of the membrane spanning proteins and is presumed to be involved in the regulation of cell growth.
  • expression vectors encoding HTMPN are electroporated into highly motile cell lines, such as U-937 (ATCC CRL 1593), HEL 92.1.7 (ATCC TIB 180) and MAC 10, and the motility of the electroporated and control cells are compared.
  • Methods for the design and construction of an expression vector capable of expressing HTMPN in the desired mammalian cell line(s) chosen are well known to the art.
  • HTMPN Assays for examining the motility of cells in culture are known to the art (cf Miyake, M. et al. (1991) J. Exp. Med. 174:1347-1354 and Ikeyama, S. et al. (1993) J. Exp. Med. 177:1231-1237).
  • Increasing the level of HTMPN in highly motile cell lines by transfection with an HTMPN expression vector inhibits or reduces the motility of these cell lines, and the amount of this inhibition is proportional to the activity of HTMPN in the assay.
  • the activity of HTMPN may be measured using an assay based upon the property of MPs to support in vitro proliferation of fibroblasts and tumor cells under serum-free conditions.
  • HTMPN or biologically active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (See, e.g., Bolton et al. (1973) Biochem. J. 133:529).
  • Bolton-Hunter reagent See, e.g., Bolton et al. (1973) Biochem. J. 133:529).
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HTMPN, washed, and any wells with labeled HTMPN complex are assayed. Data obtained using different concentrations of HTMPN are used to calculate values for the number, affinity, and association of HTMPN with the candidate molecules.
  • XI Functional Assays
  • HTMPN function is assessed by expressing the sequences encoding HTMPN at physiologically elevated levels in mammalian cell culture systems.
  • cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter. 5-10 ⁇ g of recombinant vector are transiently transfected into a human cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.
  • FCM Flow cytometry
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York NY.
  • HTMPN The influence of HTMPN on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding HTMPN and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG).
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding HTMPN and other genes of interest can be analyzed by northern analysis or microarray techniques. XII.
  • HTMPN substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • PAGE polyacrylamide gel electrophoresis
  • HTMPN amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • LASERGENE software DNASTAR
  • Methods for selection of appropriate epitopes. such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel. 1995, supra, ch. 11.)
  • oligopeptides 15 residues in length are synthesized using an ABI 431 A Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity.
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio- iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinant HTMPN is substantially purified by immunoaffinity chromatography using antibodies specific for HTMPN.
  • An immunoaffinity column is constructed by covalently coupling anti-HTMPN antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham
  • HTMPN Media containing HTMPN are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HTMPN (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody /HTMPN binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HTMPN is collected.
  • ⁇ BI/PARACEL FDF A Fast Data Finder useful in comparing and annotating Perkin-Elmer Applied Biosystems, Mismatch ⁇ 50% amino acid or nucleic acid sequences. Foster City, CA; Paracel Inc., Pasadena, CA.
  • Phred A base-calling algorithm that examines automated Ewing, B. et al. ( 1998) Genome sequencer traces with high sensitivity and probability. Res. 8: I 75- 185; Ewing, B. and P. Green ( 1998) Genome Res. 8: 186- 194.
  • Motifs A program that searches amino acid sequences for patterns Bairoch et al. supra; Wisconsin that matched those defined in Prosite Package Program Manual, version 9, page M51 -59, Genetics Computer Group, Madison, WI.

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Abstract

L'invention porte sur des protéines transmembranaires humaines et sur des polynucléotides identifiant et codant ces protéines. L'invention porte également sur des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes, ainsi que sur des procédés de diagnostic, de traitement ou de prévention des maladies associées à l'expression des protéines transmembranaires humaines.
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